Vector for integration of heterologous sequences into poxviral genomes

ABSTRACT

A new DNA vector is disclosed comprising a nucleic acid sequence useful for inserting heterologous sequences into the genome of poxviruses by homologous recombination. Also disclosed are recombinant poxviruses carrying heterologous coding sequences transferred by the DNA vector.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of copending application Ser.No. 09/980,029 filed 26 Dec. 2001.

FIELD OF THE INVENTION

[0002] The present invention provides a new DNA vector comprising anucleic acid sequence useful for inserting heterologous sequences intothe genome of poxviruses by homologous recombination. The presentinvention relates also, inter alia, to recombinant poxviruses carryingheterologous coding sequences transferred by the vector according to thepresent invention.

BACKGROUND OF THE INVENTION

[0003] The successful worldwide eradication of smallpox via vaccinationwith live Orthopoxvirus, such as Vaccinia virus strain Western Reserve,Copenhagen or Ankara, stimulated in the early 80's research to studypoxvirus s in closer detail. Subsequently, said poxviruses weredeveloped to well understood and easy-to-handle virus vectors orresearch tools, respectively (Moss, 1996). Today poxvirus vectors areused in various fields e.g. as expression vector or for the developmentof vaccines and therapeutic substances. The main reasons for the highacceptance of poxvirus vectors are the following promising features:Firstly, the vector viruses are easy to manipulate, are highly stableand cheap to manufacture. Secondly, said vector virus can accommodatelarge amounts of heterologous DNA and has proved to be a versatileexpression vector. Thirdly, said vector virus is easily administered invivo and has succeeded in stimulating humoral and cellular immuneresponses. Accordingly, its use as a recombinant vaccine for protectiveimmunization against infectious disease or cancer made poxvirus vectorsparticularly attractive. Especially, Vaccinia virus, the best-knownmember of the Orthopoxvirus family, has been successfully used asrecombinant vaccine to protect against diseases in a large variety ofanimal models (Carroll et al., 1997; Sutter et al., 1994a).

[0004] To develop and establish recombinant vaccinia viruses severalinsertion sites have been used. The most prominent insertion site of thevaccinia genome is the locus of the viral thymidine-kinase (tk) gene(Mackett et al., 1982). However, also other non-essential genes, such asthe viral hemagglutinin and ribonucleotide reductase genes (Shida et al.1987, Howley et al. 1996) or the naturally occurring deletion site II orIII have been used to insert heterologous DNA sequences into the genomeof vaccinia virus (Sutter et al., 1994a). Construction of recombinantvector viruses carrying several heterologous genes or severalimmunogenic epitopes becomes more and more of general interest.Accordingly, there is a high need to identify further sites in the virusgenome, which are suitable for insertion of further heterologous DNAsequences.

[0005] Insertion of heterologous DNA sequences into a poxviral genomebears the risk to destroy regions essential for the virus propagationdue to a lack of complete understanding of the poxviral life cycle.Although the sequence information of several poxvirus genomes (Goebel etal. 1990; Antoine et al. 1998) is available, the function of mostproteins encoded by the identified open reading frames is not known.Accordingly, it is still a complicated challenge to identify sites inthe genome, which are suitable to stably take up heterologous DNAwithout destroying any sequences essential for viral replication andpropagation.

OBJECT OF THE INVENTION

[0006] It is thus an object of the present invention to identify a newinsertion site in the poxviruses genome and provide vectors suitable todirect the integration of heterologous DNA sequences into said insertionsite.

DESCRIPTION OF THE INVENTION

[0007] To achieve the foregoing and other objects, the present inventionprovides a vector comprising a nucleic acid sequence according to SeqIDNo. 1 or its complementary strand. The nucleic acid sequence accordingto SeqID No. 1 is highly homologous with parts of the genomic sequencesof a poxvirus genome. Due to this homology the nucleic acid sequenceaccording to the present invention is capable to initiate homologousrecombination between said sequence and the corresponding genomicsequences of poxviruses. Thus, the present invention provides a meansuseful to direct integration of DNA sequences into the genome ofdifferent orthopoxviruses, preferably into the genome of modifiedvaccinia virus Ankara (MVA), but also of further related orthopoxvirusessuch as, e.g., Vaccinia virus strain Western Reserve or Copenhagen.

[0008] According to a preferred embodiment the nucleic acid sequence ofthe present invention is derived from modified vaccinia Ankara virus(MVA), especially from MVA, which has been isolated and deposited onJan. 27th, 1994 according to the Budapest Treaty at the EuropeanCollection of Animal Cell Cultures (Salisbury, UK) under Deposit No.:V94012707.

[0009] The present invention further provides a vector comprisingnucleic acid sequences, which hybridize under stringent conditions tothe sequences according to SegIDNo: 1 or its complementary strand. Inthe context of this invention the term “vector” is understood as DNAvehicles of circular structure, such as plasmids, cosmids or artificialchromosomes. Said vector comprises in addition to the desired nucleicacid sequence regulatory seq fences, selective marker genes andreplicons enabling the autonomous replication of the vector. Hence, thevector according to the present invention can easily be amplified in andisolated from unicellular host organism. Furthermore, the term “understringent conditions” defines parameters according to standard protocols(Sambrook et al., 1989), such as reaction temperature, formamide contentor salt concentrations, which allow hybridization of DNA-DNA sequenceswith a homology about and above 70%. As described above, these sequencesalso hybridize to the corresponding sequence of the poxvirus genome andare thus, particularly, useful to integrate heterologous sequences intoa genome of orthopoxviruses.

[0010] Additionally, the present invention provides a vector comprisingfragments of the above-mentioned nucleic acid sequence. These fragmentscomprise consecutive base pairs of said nucleic acid sequence and arealso useful to integrate into the poxviral genome by homologousrecombination. The length of said fragments is variable and fragmentswith only 30 base pairs being homologous to corresponding parts of thepoxvirus genome are already sufficient to initiate recombination events.However, to increase the efficiency of homologous recombination betweenthe poxvirus genome and the fragments as used in the present invention,said fragments are preferably about and above 200 base pairs in length,more preferably about and above 300 or 500 base pairs in length.

[0011] To initiate homologous recombination the vector according to thepresent invention and a wild type poxvirus are introduced into a hostcell. During replication of the poxvirus genome homologous recombinationbetween the nucleic acid sequence inserted into the vector and thecorresponding sequences of the poxvirus genome occurs. Since homologousrecombination events occur only with a statistical probability of 1:10 3to 1:10 4 any resulting recombinant poxvirus needs to be isolated. Forthis, e.g. a marker gene with functionally associated regulatory elementis inserted into a cloning site of the nucleic acid sequence included inthe vector. After homologous recombination the resulting recombinantpoxviruses are isolated by screening for expression of said marker geneor by selection for the expression of a dominant-selection marker gene,respectively.

[0012] According to a further embodiment the vector of the presentinvention is particularly useful for insertion of a desired heterologouscoding sequence into a poxviral genome. The term “heterologous” is usedin the context of this invention for any combination of nucleic acidsequences that is not normally found intimately associated in nature.The heterologous genes according to the present invention are preferablyselected from the group of marker genes, therapeutic genes, such asanti-viral genes, anti-tumor genes, cytokine or chemokine genes, suicidegenes, but also from host range genes or immunogenic epitopes. Forinsertion and/or expression of a desired heterologous coding sequencesinto a poxviral genome said heterologous coding sequence is inserted ata cloning site within the nucleic acid sequence.

[0013] In general, a cloning site is a restriction enzyme recognitionsite. According to the present invention the preferred cloning site forthe insertion of heterologous sequences is the restriction enzymerecognition site of the EcoRI enzyme. This EcoRl site is unique in thenucleic acid sequence of the present invention, and is located betweenthe two ORFs included in said nucleic acid sequence (FIG. 1). Besidethis, any further restriction enzyme recognition site, which is locatedin the non-coding regions between said two ORFs can be used as cloningsite. Surprisingly, also any cloning site located in one of the ORFs canbe used for the insertion of heterologous sequences. Particularly, theinventors found that the destruction of said ORFs by such insertion intothe ORFs does not hamper the viral life cycle or replication efficiency,respectively. Additionally, it was found by the inventors that the useof fragments according to the invention, which are incorporated in thevector to initiate homologous recombination, likewise did not interferewith viral propagation or replication efficiency.

[0014] Generally, heterologous sequences to be integrated into a viralgenome by homologous recombination are flanked on both ends by sequencesbeing homologous to corresponding sequences of the viral genome.However, the present invention also includes vectors wherein theheterologous sequence is only flanked on one side by the above-mentionednucleic acid sequence. According to the present invention also vectorscomprising only one fragment and a desired heterologous sequence areuseful to insert said heterologous sequence into a poxvirus genome.

[0015] To guarantee expression of an inserted heterologous codingsequence at least one transcriptional control element is additionallyinserted into the cloning sites. This transcriptional control element isin functional association with the heterologous coding sequence, therebycontrolling and/or allowing its expression. According to a furtherpreferred embodiment of the present invention the transcription controlelement is derived from a poxvirus and/or is a consensus sequence of apoxvirus derived transcription control element.

[0016] According to still a further embodiment of the present inventionthe vector comprises at least two recombinogenic sequences, which flankone or more heterologous coding sequences, particularly sequencesencoding e.g. a marker or a host range gene, and/or the transcriptioncontrol element(s) inserted into the cloning site. The term“recombinogenic sequences” describes nucleic acid sequences, which, dueto their similar or nearly identical structure, are capable to deleteany sequence between said recombinogenic sequences by intragenomichomologous recombination. Accordingly, the sequences flanked by saidrecombinogenic nucleic acid sequences are only transiently inserted intothe viral genome and are, subsequently, completely deleted. Thisdeletion of sequences flanked by recombinogenic sequences is ofparticular interest for the isolation of recombinant poxviruses, whichshould comprise only a heterologous coding sequence encoding atherapeutic or immunogenic gene, but no further marker or host rangegene. For this, the marker gene, the host range gene and/or eventuallyalso the transcription control element(s), but not the desiredheterologous coding sequence, e.g. a therapeutic gene or immunogenicepitopes, are flanked by such recombinogenic sequences. After isolationof the recombinant virus, which is performed under a selection pressureupon the marker gene or the host range gene, the selection pressure isremoved and thus, intragenomic homologous recombination to delete themarker or host range gene is allowed.

[0017] The present invention, furthermore, provides a recombinantpoxvirus comprising in its genome the nucleic acid sequence transferredby the vector according to the present invention. Most preferably, thisrecombinant poxvirus is a recombinant MVA virus.

[0018] A further embodiment of the invention provides a method oftreatment and/or prevention of an infectious disease or proliferativedisorder. Said method comprises infection—either in vivo or in vitro—ofa target cell population with recombinant poxviruses according to thepresent invention. Alternatively, according to this method the targetcells are transduced—either in vivo or in vitro—with the vectoraccording to the present invention and are infected, simultaneously orwith a timelag, with any orthopoxvirus, including the recombinantpoxvirus of the present invention. In this case, the poxvirus providesthe cell with the poxviral replication and transcription machinery. As aconsequence, the desired heterologous coding sequence incorporated inthe vector and controlled by a poxvirus-derived transcriptional controlelement is expressed in the target cell. Target cells, which have beentransduced or infected in vitro, can then according to the method of thepresent invention be applied to a living animal body, including a human.

[0019] The invention provides the vector, the recombinant poxvirusand/or the target cells of the present invention useful for thetreatment and/or prevention of an infectious disease or proliferativedisorder. Furthermore, the vector, the recombinant poxvirus and/or thetarget cells according to the present invention are used for theproduction of a pharmaceutical composition, especially a vaccine, whichis useful for in vivo and in vitro gene delivery and/or vaccination ofmammals including humans, as described above.

SUMMARY OF THE INVENTION

[0020] The present invention, inter alia, comprises the following aloneor in combination:

[0021] A vector for insertion of heterologous coding sequences into apoxviral genome, said vector including a nucleic acid sequencecomprising one or more elements selected from the group consisting of:

[0022] (a) the nucleic acid sequence according to SeqID No. 1 or itscomplementary strand;

[0023] (b) a nucleic acid sequence which hybridizes under stringentconditions to the sequences as defined in (a);

[0024] (c) a fragment comprising at least 30 consecutive base pairs ofthe nucleic acid sequences as defined in (a) or (b);

[0025] the vector as above wherein the nucleic acid sequence is derivedfrom a modified vaccinia Ankara virus (MVA);

[0026] the vector as above wherein additionally at least onetranscriptional control element is included into at least one cloningsite of said nucleic acid sequence;

[0027] the vector as above wherein the transcriptional control elementis derived from a poxvirus genome or is the consensus sequence of apoxvirus derived transcriptional control element;

[0028] the vector as above additionally comprising at least oneheterologous coding sequence, said heterologous coding sequencefunctionally associated with the transcriptional control element asabove;

[0029] the vector as above wherein the heterologous coding sequence isselected from the group of marker genes, therapeutic genes, host rangegenes and/or immunogenic epitopes;

[0030] the vector as above comprising a recombinogenic sequence, whichflanks one or more heterologous coding sequences encoding marker genes,host range genes and/or the transcriptional control element as above;

[0031] a recombinant poxvirus comprising in its genome the nucleic acidsequence transferred by the vector as above;

[0032] the recombinant poxvirus as above wherein the poxvirus is amodified vaccinia Ankara virus (MVA);

[0033] a method of introducing a heterologous sequence into poxvirusgenome comprising

[0034] (a) transduction of a host cell with the vector as above

[0035] (b) infection of said host cell with a poxvirus, and

[0036] (c) isolation of recombinant poxviruses;

[0037] a method of treatment and/or prevention of an infectious diseaseor proliferative disorder of a living animal body, including a human,comprising application to said living animal body the recombinantpoxvirus as above, and/or the vector as above, or application of saidvector with any other poxvirus;

[0038] the method as above wherein the recombinant poxvirus is derivedfrom an orthopoxvirus;

[0039] a target cell comprising the recombinant poxvirus as above and/orthe vector as above;

[0040] the vector as above, the recombinant poxvirus as above and/or thetarget cell as above for the treatment and/or prevention of aninfectious disease or proliferative disorder;

[0041] the use of the vector as above, the recombinant poxvirus as aboveand/or the tar get cell as above for the production of a medicament forthe treatment and/or prevention of an infectious disease orproliferative disorder;

[0042] a pharmaceutical composition comprising the vector as above, therecombinant poxvirus as above and/or the target cell as above, and apharmaceutical acceptable carrier and/or diluent;

[0043] a pharmaceutical composition comprising the vector as above, apoxvirus, except the recombinant poxvirus as above, and a pharmaceuticalacceptable carrier and/or diluent.

BRIEF DESCRIPTION OF THE DRAWING

[0044]FIG. 1 is a map of the polynucleotide having Seq. ID No:1 withrestriction sites xhoI.1, EcoRI, and Hind III and open reading framesORF-1 and ORF-2;

[0045]FIG. 2 is a map of plasmid pSW1 comprising the polynucleotidehaving Seq. ID No: 1.

[0046] The following example will further illustrate the presentinvention. It will be well understood by a person skilled in the artthat the provided example in no way may be interpreted in a way thatlimits the applicability of the technology provided by the presentinvention to this example, and the invention is therefore to be limitedonly by the full scope of the appended claims.

EXAMPLE 1

[0047] Construction of the Insertion Vector

[0048] To obtain sequences suitable for recombination into a poxviralgenome, a DNA fragment derived from the modified vaccinia Ankara virus(deposited according to the Budapest Treaty under Deposit No.: V94012707at the European Collection of Animal Cell Cultures in Salisbury, UK) wasamplified by conventional PCR using the following oligonucleotideprimers: A24R_1;5′- CCG AAGCTTAATGAACGCCAGAGG- 3′, SeqID No.: 2;A27L_1c;5′- AGGCTCGAGTAAGAGCGGCTATGAT- 3′, SegID No.: 3.

[0049] The oligonucleotide primers comprise, close to the 5′ end andmarked by underlining, a recognition sequence for the restrictionenzymes HindIII (SeqID No.: 2) or XhoI (SeqlD No.: 3) for subcloning ofthe resulting amplification product into a cloning vector. Accordingly,the specifically amplified sequence (SeqID No.: 1), which has amolecular weight of 1.7 kb, was subcloned SaII/HindIII into a pUC19cloning plasmid (GenBank Accession No.: X02514). The resulting plasmidwas designated pSW1 (FIG. 2).

[0050] The subcloned insert has been sequenced, and this sequence wascompared to other known sequences from vaccinia virus strainsCopenhagen, WR, and MVA. It was found that said sequence comprises partsof the sequence of the MVA-ATI region. The ATI gene of mostorthopoxviruses form a dense cytoplasmic matrix embedding maturevirions, so called inclusion bodies, which can be visualized by lightmicroscopic examination of infected cells. Proposed ATI function is toprovide higher stability and prolonged dissemination of infectious virusparticles in the general environment. Among the orthopoxviruses areseveral members including ectromelia virus, cowpox virus and racoonpoxvirus produce this typical inclusion protein with a size of 130 to160 kDa. However, other members of the orthopox genus, as e.g. vacciniavirus Western Reserve (WR), vaccinia virus Copenhagen or MVA, form nosuch inclusion bodies. This is a result of sequence deletions orframe-shift mutations leading to loss of coding sequence and resultingin a truncated ATI-homologue. For example vaccinia virus WR expresses a94 kDa ATI-homologue, while MVA and vaccinia virus strain Copenhagenexpresses no such ATI-homologue.

[0051] For the further construction of insertion vectors, a naturallyoccurring recognition site of the restriction enzyme EcoRI was used tosplit the amplified sequence into two segments. These segments serve asflanking regions (flank1, flank2), that initiate homologousrecombination with a poxvirus genome. In between these flanking regions,vaccinia virus promoter sequences—e.g. of the 7,5 promoter (7.5) and/orthe synthetic promoter (sP)—as well as multiple cloning sites for theinsertion of operably heterologous genes have been inserted. Theresulting plasmids are designated pSW-7.5-sP, pSW-7.5, pSW-sP.

[0052] Additionally an expression cassette comprising the vaccinia virushost rage gene, K1L, fused to the EGFP fusion gene (isolated from theplasmid pEGFP, Clonetech, GenBank Accession #: U76561) and the naturallyoccurring K1L promoter, was inserted between the flanking regions asdescribed above. This expression cassette is especially helpful forefficient sel action and isolation of recombinant viruses. The resultingplasmid is designated pSWkllgfp.

[0053] Generation of Recombinant Virus

[0054] For the generation of recombinant viruses 6-well tissue cultureplates with cell monolayers of about 80% confluence are used. For thegeneration of recombinant MVA permissive cells such as chicken embryofibroblasts are used. For the generation of recombinant vaccinia virusesof the strain WR African Green Monkey (Vero) cells have been used.

[0055] Firstly, the cell culture medium is discarded and the cellsoverlaid with serum-free medium containing wild-type poxvirus at amultiplicity of infection (MOI) of 0.01 (e.g. an inoculum 5×1031U(infectious units) in 1 ml medium for one well with 5×105 cells). Thismixture is incubated for 1 hour at 37° C. in 5% C02-atmosphere. Then,the inoculum is removed and the cells are washed twice with 2 ml OptiMEMper well. Subsequently, the cell monolayer is overlaid withLipofectin/plasmid DNA-mix (total volume: 1 ml) prepared as described bythe manufacturer (GIBCO BRL) and using 15=g plasmid DNA, of thepSWkllgfp. The mixture I s incubated for 5-12 hours at 37° C. in 5%C02-atmosphere. Then, the Lipofectin/plasmid DNA-mix is removed and thecells overlaid with 1.5 ml fresh medium supplemented with 10% FCS.

[0056] At 48 hours after infection, cell monolayer is detached with acell scraper and the cells and medium are transferred into 2ml-microcentrifuge tubes. The transfection harvest is stored at −20 to−80° C.

[0057] Upon transfection of the plasrnid pSWkllgfp into poxvirusinfected cells, the host range gene KIL fused to the EGFP gene wereprecisely recombined into the site of the poxvirus genome, which ishomologous with the flanking regions in the vector plasmid.

[0058] To isolate recombinant MVA viruses from non-recombinant MVAviruses a host range cell, rabbit kidney (RK)13, was infected with virusmaterial obtained from the transfection experiment as described above.Previous work had shown that MVA infection of rabbit RK13 cells resultsin an early block of viral replication characterized by impairedsynthesis of intermediate viral RNA and lacking replication viral DNA.However, this non-productive NVA infection of RK13 cells could beovercome by coexpression of the vaccinia virus host range gene K1L(Sutter et al., 1994b). Accordingly, inoculation of virus materialobtained from said transfection experiments into RK13 cultures resultedin the highly selective growth only of recombinant viruses, whichcoexpressed the KM gene. After five consecutive passages on RK13 cellsgrown in 6-well or 96-well tissue culture plates the virus MVA-KILGFPwas isolated. The absence of nonrecombinant MVA was demonstrated by PCR.

[0059] Additionally, the expression of the fused EGFP gene allowed adirect monitoring of infection with recombinant virus via GFPfluorescence. The direct monitoring is also used to identify and thenisolate recombinant Vaccinia viruses of the strain WR, which are notsensitive to a K1L selection.

References

[0060] Antoine, G. et al., 1998, The complete genomic sequence of themodified vaccinia Ankara strain: comparison with other orthopoxviruses.Virology, 5 May 10;244(2):365-96.

[0061] Carroll, M. W. et al. 1997: Highly attenuated modified vacciniavirus Ankara (MVA) as an effective recombinant vector: A murine tumormodel. Vaccine 15,387.

[0062] Fenner, F. et al. 1988: Smallpox and its eradication. WorldHealth Organisation, Geneva.

[0063] Goebel, S J. et al., 1990, The complete DNA sequence of vacciniavirus. 15 Virology, November;179(l):247-66, 517-63.

[0064] Howley, P M. et al., 1996, A vaccinia virus transfer vector usinga GUS reporter gene inserted into the 14L locus. Gene, June26;172(2):233-7.

[0065] Mackett, M. et al. 1982: Vaccinia virus: a selectable eukaryoticcloning and expression vector. Proc. Natl. Acad. Sci. USA 79, 7415.

[0066] Moss, B. 1996: Genetically engineered poxviruses for recombinantgene expression, vaccination, and safety. Proc. Natl. Acad. Sci. USA93,11341.

[0067] Sambrook, et al., 1989, Molecular Cloning: a laboratory manual,Cold Spring Harbor Laboratory Press publication, New York.

[0068] Shida, H. et al., 1987, Effect of the recombinant vacciniaviruses that express 30 HTLV-I envelope gene on HTLV-I infection. EMBOJ. November;6(11):3379-84.

[0069] Sutter, G. et al. 1994 a: A recombinant vector derived from thehost range-restricted and highly attenuated MVA strain of vaccinia virusstimulates protective immunity in mice to influenza virus. Vaccine 12,1032.

[0070] Sutter, G. et al. 1994b: Stable expression of the vaccinia virusK1L gene in rabbit cells complements the host range defect of a vacciniavirus mutant. J. Virol. 68, 4109.

1 3 1 1736 DNA Modified vaccinia Ankara virus 1 ctcgagtaag agcggctatgatatctctgg ctaaaaagat tgatgttcag actggacggc 60 gtccatatga gtaacttaactcttttgtta attaaaagta tattcaaaaa atgagttata 120 taaatggcga acattataaatttatggaac ggaattgtac caacggttca agatgttaat 180 gttgcgagca ttactgcgtttaaatctatg atagatgaaa catgggataa aaaaatcgaa 240 gcaaatacat gcatcagtagaaaacataga aacattattc acgaagttat tagggacttt 300 atgaaagcat atcctaaaatggacgagaat agaaaatctc cattaggagc tccaatgcaa 360 tggctaacac aatattatattttaaagaat gaatatcata agaccatgct agcgtatgat 420 aatggatcat tgaatacaaaatttaaaacg ttaaacattt atatgattac taacgttggt 480 caatatattt tatatatagtattttgtata atatctggta agaatcacga tggtactcct 540 tatatatacg attctgagataacgagcaat gataaaaatc ttattaatga gcgtatcaag 600 tatgcatgta agcaaatattacacggtcaa ttaactatag ctctgagaat tagaaataaa 660 ttcatgttta taggatcacccatgtattta tggtttaacg taaacggatc acaggtatat 720 cacgacatat atgatcgtaatgccggtttt cataataaag agataggtag actactatac 780 gcatttatgt actatctatctatctataag tggtagattt ttgaatgatt tcgcactatt 840 aaagtttacg tatttaggagaatcctggac atttagtttg agtgttcctg aatatatatt 900 atatggttta ggatattctgttttcgatac tattgaaaaa tttagcaatg atgctatact 960 cgtttatgtt agaacaaacaatagaaatgg atatgattat gttgagttta ataaaaaagg 1020 aattgctaag gtgacagaagcctaaacccg ataacgataa gcgaattcat gctataagac 1080 gcatgaaggc tgaacgtgaaatcgctcgta aaaactgcgg aggtaaccca tgcgaacgtg 1140 aattgaaatc tgaacgtagtaacgtgaaga ggttggaata tcaactagat gctgagaaag 1200 aaaaagttaa gttctacaaaagagaactag aacgtgatcg gtatctttct agtagatatc 1260 ttacctcttc ttcagatccacatgagaaac cattaccaaa ttatacattt cctcgcatta 1320 aaaatgtatc tccgttgacaactgaggcta caggttctgt agaagtagca cctccatcca 1380 cagacgttac cgaaccgattagtgatgtga caccatcggt ggatgtcgaa ccagaacatc 1440 ccccagcttt ctgaatatcagacttcagta tcccaagtag cagttacacc tccaccaaaa 1500 cctgaaactc cacagattttcgaatatcag acgtccgatt ctatagttaa caatccacgc 1560 ccattttata attcggatctcgaatttgat gatattgata tgtatctact accaaactag 1620 aatattacac cagaaaagacggcttgagat caactttatc taatggttta taaaacgaag 1680 gaggccttcg ttcgaaatctaatttgactt ttacgcctct ggcgttcatt aagctt 1736 2 24 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide primer 2ccgaagctta atgaacgcca gagg 24 3 25 DNA Artificial Sequence Descriptionof Artificial Sequence oligonucleotide primer 3 aggctcgagt aagagcggctatgat 25

What is claimed is:
 1. A nucleic acid sequence including at least onecloning site and selected from the group consisting of: (a) a nucleicacid sequence according to Seq ID No. 1 or its complementary strand, (b)a nucleic acid sequence that hybridizes under stringent conditions tothe nucleic acid sequence as defined in (a), and (c) a fragmentcomprising at least about 200 consecutive base pairs of the nucleic acidsequence as defined in (a) or in (b).
 2. A vector for insertion of aheterologous sequence into the ATI region of an orthopoxviral genome,said vector including a nucleic acid sequence selected from the groupconsisting of: (a) a nucleic acid sequence according to Seq ID No. 1 orits complementary strand, (b) a nucleic acid sequence that hybridizesunder stringent conditions to the nucleic acid sequence as defined in(a), and (c) a fragment comprising at least about 200 consecutive basepairs of the nucleic acid sequence as defined in (a) or in (b).
 3. Thevector according to claim 2 wherein the nucleic acid sequence includesat least one cloning site.
 4. The vector defined in claim 3 whereinadditionally at least one transcriptional control element is included inthe cloning site of said nucleic acid sequence.
 5. The vector defined inclaim 3 wherein the cloning site is the restriction site EcoRI.
 6. Thevector defined in claim 4 wherein the at least one transcriptionalcontrol element is obtained from a poxvirus genome or is a consensussequence from a poxvirus genome.
 7. The vector defined in claim 2further comprising at least one heterologous sequence, said heterologoussequence functionally associated with a transcriptional control elementthereof.
 8. The vector defined in claim 7 wherein the heterologoussequence is selected from the group consisting of marker genes,therapeutic genes, host range genes and genes encoding immunogenicepitopes.
 9. The vector defined in claim 7 comprising a recombinogenicsequence, which flanks one or more heterologous sequences encodingmarker genes, host range genes, and or a transcriptional elementthereof.
 10. A recombinant orthopoxvirus having an ATI gene, comprisingin its ATI gene region the nucleic acid sequence defined in claim 1 andan inserted heterologous sequence.
 11. The recombinant orthopoxvirusdefined in claim 10 wherein the orthopoxvirus is selected from the groupconsisting of a modified vaccinia Ankara virus, vaccinia virus WesternReserve, and vaccinia virus Copenhagen.
 12. The recombinantorthopoxvirus defined in claim 11 wherein the orthopoxvirus is themodified vaccinia Ankara virus.
 13. A method of introducing aheterologous sequence into the ATI gene region of an orthopoxvirushaving an ATI gene to obtain a recombinant orthopoxvirus which comprisesthe steps of: (a) transducing a host cell with a vector as defined inclaim 2 comprising at least one heterologous sequence; (b) infectingsaid host cell with an orthopoxvirus having an ATI gene; (c) insertingthe heterologous sequence into an insertion site of the ATI gene of theorthopoxvirus by homologous recombination between the nucleic acidsequence and a corresponding genomic sequence of the orthopoxvirus toobtain a recombinant orthopoxvirus; and (d) isolating said recombinantorthopoxvirus.
 14. The method of introducing a heterologous sequenceinto the gene region of the orthopoxvirus having an ATI gene defined inclaim 13 wherein according to step (b) the orthopoxvirus is modifiedvaccinia Ankara virus.
 15. A target cell comprising the recombinantorthopoxvirus having an ATI gene defined in claim
 10. 16. A target cellcomprising the vector defined in claim
 2. 17. A pharmaceuticalcomposition for effecting an immune response against an infectiousdisease or a proliferative disorder which consists essentially of atherapeutically effective amount of the recombinant poxvirus as definedin claim 10 and in a form capable of producing an immune responseagainst an infectious disease or a proliferative disorder in combinationwith a pharmaceutically acceptable inert carrier or diluent.
 18. Amethod of effecting an immune response against an infectious disease ora proliferative disorder in an animal subject which comprises the stepof administering to said subject a therapeutically effective amount ofthe pharmaceutical composition defined in claim 17.